Sickle cell disease (SCD) and Cooley's anemia (CA) are disorders ameliorated by continued expression of fetal hemoglobin; first shown in patients with continued expression of fetal globin due to deletional or nondeletional hereditary persistence of fetal hemoglobin (HFPH). However, others express high fetal globin in the absence of mutation, and therapeutic agents, such as hydroxyurea, induce expression of Hb F. If the rate-limiting factors involved in the regulation of fetal hemoglobin were elucidated, targeted drug discovery could succeed in developing agents with this specific effect. Our proposal focuses on three areas: 1) identify new candidate modifiers using unbiased, genome-wide microarray-based expression profiling to define genes critical to induction of Hb F in cultured human erythroid progenitors. The candidates will be assessed for identification of genes that fall into several categories, including transcription factors, signaling and chromatin remodeling molecules. 2) examine suspected regulators of Hb F expression as well as a subset of differentially-expressed transcripts comparing cord to adult erythroid cultures for functionally relevant genetic variation associated with elevated Hb F levels. Known regulators (KLFs, NFE-4 and soluble guanylate cyclase) and the most promising of the candidate transactivators identified by expression profiling will be explored to identify putative coding region and proximal promoter sequence allelic variants using both public databases and SNP discovery methods. Those with allele frequencies of 10% or more in a small group of SCD patients, will be tested in sequential cohorts of adult and pediatric patients with SCD with and without elevated Hb F. Significant QTL loci will then be examined in a group of parent-child trios and followed with a third population of SCD patients. 3) functional validation of candidate modifiers will be accomplished by knockdown technology employing morpholino antisense oligos and/or interference RNA (iRNA) using K562 cells containing a fetal globin promoter-driven GFP readout from a beta-like globin gene cluster BAC. We are confident that our strategy to identify critical regulatory molecules for Hb F regulation and our ability to quickly and reliably screen for candidate gene variants will have significant impact on the development of therapeutic intervention strategies for patients with SCD and CA.